This report was created with:

R version 4.0.2 (2020-06-22)

Platform: x86_64-apple-darwin17.0 (64-bit)

locale: en_US.UTF-8||en_US.UTF-8||en_US.UTF-8||C||en_US.UTF-8||en_US.UTF-8

attached base packages: stats, graphics, grDevices, utils, datasets, methods and base

other attached packages: pander(v.0.6.3), cbmbtools(v.0.0.0.9034), vegan(v.2.5-6), lattice(v.0.20-41), permute(v.0.9-5), ggpubr(v.0.4.0), knitr(v.1.29), scales(v.1.1.1), gtools(v.3.8.2), tidyselect(v.1.1.0), forcats(v.0.5.0), stringr(v.1.4.0), dplyr(v.1.0.1), purrr(v.0.3.4), readr(v.1.3.1), tidyr(v.1.1.1), tibble(v.3.0.3), ggplot2(v.3.3.2) and tidyverse(v.1.3.0)

loaded via a namespace (and not attached): httr(v.1.4.2), jsonlite(v.1.7.0), splines(v.4.0.2), carData(v.3.0-4), modelr(v.0.1.8), assertthat(v.0.2.1), blob(v.1.2.1), cellranger(v.1.1.0), yaml(v.2.2.1), pillar(v.1.4.6), backports(v.1.1.8), glue(v.1.4.1), digest(v.0.6.25), ggsignif(v.0.6.0), rvest(v.0.3.6), colorspace(v.1.4-1), Matrix(v.1.2-18), htmltools(v.0.5.0), pkgconfig(v.2.0.3), broom(v.0.7.0), haven(v.2.3.1), openxlsx(v.4.1.5), rio(v.0.5.16), mgcv(v.1.8-31), generics(v.0.0.2), car(v.3.0-9), ellipsis(v.0.3.1), withr(v.2.2.0), cli(v.2.0.2), magrittr(v.1.5), crayon(v.1.3.4), readxl(v.1.3.1), evaluate(v.0.14), fs(v.1.5.0), fansi(v.0.4.1), nlme(v.3.1-148), MASS(v.7.3-51.6), rstatix(v.0.6.0), xml2(v.1.3.2), foreign(v.0.8-80), tools(v.4.0.2), data.table(v.1.13.0), hms(v.0.5.3), lifecycle(v.0.2.0), munsell(v.0.5.0), reprex(v.0.3.0), cluster(v.2.1.0), zip(v.2.0.4), compiler(v.4.0.2), rlang(v.0.4.7), grid(v.4.0.2), rstudioapi(v.0.11), rmarkdown(v.2.3), codetools(v.0.2-16), gtable(v.0.3.0), abind(v.1.4-5), DBI(v.1.1.0), curl(v.4.3), R6(v.2.4.1), lubridate(v.1.7.9), stringi(v.1.4.6), parallel(v.4.0.2), Rcpp(v.1.0.5), vctrs(v.0.3.2), dbplyr(v.1.4.4) and xfun(v.0.16)

I. 16S rRNA gene amplicon quantification

Hypothesis testing - 16S rRNA gene amplicon quantification


    Kruskal-Wallis rank sum test

data:  Gene_16S_copies_per_mL by Sample_Type
Kruskal-Wallis chi-squared = 29.276, df = 6, p-value = 5.394e-05


    Pairwise comparisons using Wilcoxon rank sum exact test 

data:  ddpcr$Gene_16S_copies_per_mL and ddpcr$Sample_Type 

              NTC     Iso_Ctrl PBS     Syringe_Rinse Homog_Ctrl BAL    
Iso_Ctrl      0.64000 -        -       -             -          -      
PBS           0.58947 0.40000  -       -             -          -      
Syringe_Rinse 0.42353 0.03333  0.40000 -             -          -      
Homog_Ctrl    0.58947 0.16667  0.42353 0.40000       -          -      
BAL           0.22238 0.00839  0.31818 0.27082       0.90909    -      
Lung          0.00839 0.00262  0.07955 0.00839       0.07955    0.00023

P value adjustment method: BH 

II. Quality Checks - Confirmation of Sufficient Reads

2.1 MiSeq Reads by Sample Type

2.2 OTU Counts by Sample Type

III. Principal Component Analysis

3.1 Lung Samples vs. Sampling Controls


Call:
adonis(formula = adonis.hel.df.wbn ~ otu.df.lungsamp.sampctrls$RA_Groups,      permutations = 10000, method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                                    Df SumsOfSqs MeanSqs F.Model      R2
otu.df.lungsamp.sampctrls$RA_Groups  2    3.8879 1.94394  2.3806 0.16554
Residuals                           24   19.5980 0.81658         0.83446
Total                               26   23.4859                 1.00000
                                       Pr(>F)    
otu.df.lungsamp.sampctrls$RA_Groups 9.999e-05 ***
Residuals                                        
Total                                            
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Call:
adonis(formula = adonis.hel.bl ~ adonis.otudf.bl$Sample_Type,      permutations = 10000, method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                            Df SumsOfSqs MeanSqs F.Model      R2    Pr(>F)    
adonis.otudf.bl$Sample_Type  1    2.4484  2.4484  3.1334 0.15563 9.999e-05 ***
Residuals                   17   13.2838  0.7814         0.84437              
Total                       18   15.7323                 1.00000              
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Call:
adonis(formula = adonis.hel.wn ~ adonis.otudf.wn$RA_Groups, permutations = 10000,      method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                          Df SumsOfSqs MeanSqs F.Model      R2 Pr(>F)    
adonis.otudf.wn$RA_Groups  1    2.4698 2.46976  3.3064 0.18061  4e-04 ***
Residuals                 15   11.2046 0.74697         0.81939           
Total                     16   13.6744                 1.00000           
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Call:
adonis(formula = adonis.hel.bn ~ adonis.otudf.bn$Organ, permutations = 10000,      method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                      Df SumsOfSqs MeanSqs F.Model      R2 Pr(>F)
adonis.otudf.bn$Organ  1    0.9148 0.91483 0.99522 0.05856  0.463
Residuals             16   14.7076 0.91922         0.94144       
Total                 17   15.6224                 1.00000       

3.2 Lung Samples v. Isolation Controls


Call:
adonis(formula = otu.good.lungsamp.isoctrls_hel ~ otu.df.lungsamp.isoctrls$Sample_Type,      permutations = 10000, method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                                     Df SumsOfSqs MeanSqs F.Model     R2
otu.df.lungsamp.isoctrls$Sample_Type  3    5.0693  1.6898  2.0376 0.1846
Residuals                            27   22.3910  0.8293         0.8154
Total                                30   27.4603                 1.0000
                                        Pr(>F)    
otu.df.lungsamp.isoctrls$Sample_Type 9.999e-05 ***
Residuals                                         
Total                                             
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Call:
adonis(formula = adonis.hel.il ~ adonis.otudf.il$RA_Groups, permutations = 10000,      method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                          Df SumsOfSqs MeanSqs F.Model      R2    Pr(>F)    
adonis.otudf.il$RA_Groups  1    2.6804 2.68040  3.4196 0.15253 9.999e-05 ***
Residuals                 19   14.8927 0.78383         0.84747              
Total                     20   17.5731                 1.00000              
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Call:
adonis(formula = adonis.hel.ib ~ adonis.otudf.ib$RA_Groups, permutations = 10000,      method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                          Df SumsOfSqs MeanSqs F.Model      R2  Pr(>F)  
adonis.otudf.ib$RA_Groups  1    1.2087 1.20866  1.3141 0.06165 0.07309 .
Residuals                 20   18.3957 0.91978         0.93835          
Total                     21   19.6044                 1.00000          
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

3.3 Lung Samples vs. Sequencing Controls


Call:
adonis(formula = otu.good.lungsamp.seqctrls_hel ~ otu.df.lungsamp.seqctrls$Sample_Type,      permutations = 10000, method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                                     Df SumsOfSqs MeanSqs F.Model      R2
otu.df.lungsamp.seqctrls$Sample_Type  3     7.919 2.63959  3.2847 0.16193
Residuals                            51    40.984 0.80361         0.83807
Total                                54    48.903                 1.00000
                                        Pr(>F)    
otu.df.lungsamp.seqctrls$Sample_Type 9.999e-05 ***
Residuals                                         
Total                                             
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Call:
adonis(formula = adonis.hel.sl ~ adonis.otudf.sl$RA_Groups, permutations = 10000,      method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                          Df SumsOfSqs MeanSqs F.Model      R2    Pr(>F)    
adonis.otudf.sl$RA_Groups  1     4.146  4.1459  5.1613 0.10717 9.999e-05 ***
Residuals                 43    34.541  0.8033         0.89283              
Total                     44    38.687                 1.00000              
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Call:
adonis(formula = adonis.hel.sb ~ adonis.otudf.sb$RA_Groups, permutations = 10000,      method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                          Df SumsOfSqs MeanSqs F.Model      R2 Pr(>F)    
adonis.otudf.sb$RA_Groups  1     2.069 2.06931  2.3933 0.05159  5e-04 ***
Residuals                 44    38.044 0.86463         0.94841           
Total                     45    40.113                 1.00000           
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

3.4 Lung Samples vs. Tongue


Call:
adonis(formula = otu.good.tong.lungsamp_hel ~ otu.df.tong.lungsamp$Sample_Type,      permutations = 10000, method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                                 Df SumsOfSqs MeanSqs F.Model      R2    Pr(>F)
otu.df.tong.lungsamp$Sample_Type  2    3.9663 1.98317  2.6355 0.12772 9.999e-05
Residuals                        36   27.0894 0.75248         0.87228          
Total                            38   31.0557                 1.00000          
                                    
otu.df.tong.lungsamp$Sample_Type ***
Residuals                           
Total                               
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Call:
adonis(formula = adonis.hel.tl ~ adonis.otudf.tl$Sample_Type,      permutations = 10000, method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                            Df SumsOfSqs MeanSqs F.Model      R2 Pr(>F)  
adonis.otudf.tl$Sample_Type  1     1.039 1.03903  1.5005 0.05265 0.0119 *
Residuals                   27    18.696 0.69244         0.94735         
Total                       28    19.735                 1.00000         
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

Call:
adonis(formula = adonis.hel.tb ~ adonis.otudf.tb$Sample_Type,      permutations = 10000, method = "euclidean") 

Permutation: free
Number of permutations: 10000

Terms added sequentially (first to last)

                            Df SumsOfSqs MeanSqs F.Model      R2    Pr(>F)    
adonis.otudf.tb$Sample_Type  1    2.6012 2.60116  3.2809 0.10489 9.999e-05 ***
Residuals                   28   22.1989 0.79282         0.89511              
Total                       29   24.8001                 1.00000              
---
Signif. codes:  0 '***' 0.001 '**' 0.01 '*' 0.05 '.' 0.1 ' ' 1

3.5 Lung vs. Tongue vs. Negative Controls

IV. Diversity Indices

4.1 Alpha Diversity - Rarified Richness

  Tukey multiple comparisons of means
    95% family-wise confidence level

Fit: aov(formula = tukey_otu_df[, "Unique_Otus_per_1k_reads"] ~ tukey_otu_df[, "Alpha_Div_Groups"])

$`tukey_otu_df[, "Alpha_Div_Groups"]`
                                           diff        lwr         upr
Isolation Control-BAL                -10.612291 -27.006526   5.7819429
Lung-BAL                              25.049970   7.457534  42.6424053
Sampling Control-BAL                  -2.528455 -20.690372  15.6334619
Sequencing Control-BAL               -13.515401 -27.202081   0.1712778
Lung-Isolation Control                35.662261  18.778539  52.5459833
Sampling Control-Isolation Control     8.083837  -9.392476  25.5601491
Sequencing Control-Isolation Control  -2.903110 -15.666004   9.8597843
Sampling Control-Lung                -27.578425 -46.183380  -8.9734692
Sequencing Control-Lung              -38.565371 -52.834721 -24.2960216
Sequencing Control-Sampling Control  -10.986946 -25.952766   3.9788736
                                         p adj
Isolation Control-BAL                0.3746804
Lung-BAL                             0.0014826
Sampling Control-BAL                 0.9950044
Sequencing Control-BAL               0.0545862
Lung-Isolation Control               0.0000011
Sampling Control-Isolation Control   0.6949822
Sequencing Control-Isolation Control 0.9684857
Sampling Control-Lung                0.0008519
Sequencing Control-Lung              0.0000000
Sequencing Control-Sampling Control  0.2510366

4.2 Alpha Diversity - Shannon Diversity Index

  Tukey multiple comparisons of means
    95% family-wise confidence level

Fit: aov(formula = tukey_otu_df[, "Shannon"] ~ tukey_otu_df[, "Alpha_Div_Groups"])

$`tukey_otu_df[, "Alpha_Div_Groups"]`
                                           diff        lwr        upr     p adj
Isolation Control-BAL                -0.9947478 -1.8514868 -0.1380087 0.0147639
Lung-BAL                              0.6346623 -0.2846930  1.5540176 0.3098538
Sampling Control-BAL                 -0.5859416 -1.5350572  0.3631740 0.4234102
Sequencing Control-BAL               -1.2226705 -1.9379166 -0.5074244 0.0000876
Lung-Isolation Control                1.6294101  0.7470911  2.5117290 0.0000205
Sampling Control-Isolation Control    0.4088062 -0.5044807  1.3220931 0.7201786
Sequencing Control-Isolation Control -0.2279227 -0.8948932  0.4390477 0.8732743
Sampling Control-Lung                -1.2206039 -2.1928720 -0.2483357 0.0067338
Sequencing Control-Lung              -1.8573328 -2.6030284 -1.1116372 0.0000000
Sequencing Control-Sampling Control  -0.6367289 -1.4188211  0.1453633 0.1637833

4.3 Beta Diversity - Bray-Curtis dissimilarity Index within Sample Type

[1] 87  2

    Pairwise comparisons using Wilcoxon rank sum test with continuity correction 

data:  bray_dist_lbtce_long_mut_filt_uniq$BC_Index and bray_dist_lbtce_long_mut_filt_uniq$Sample_Type 

       BAL     Blank   Cecum   Lung  
Blank  0.2730  -       -       -     
Cecum  < 2e-16 < 2e-16 -       -     
Lung   6.3e-09 7.4e-06 < 2e-16 -     
Tongue < 2e-16 8.5e-13 < 2e-16 0.0053

P value adjustment method: BH 

4.4 Bray-Curtis dissimilarity - Lung Samples vs. Matched Tongue Samples


    Wilcoxon rank sum exact test

data:  BC_Index by Comparison_Type
W = 85, p-value = 0.0004114
alternative hypothesis: true location shift is not equal to 0

V. Relative Abundance

5.1 Ordered by Lung OTUs

5.2 Ordered by Pooled Negative Control OTUs

5.3 Ordered by Tongue OTUs